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RESEARCH ARTIC LE Open Access
Uncharacterized conserved motifs outside the
HD-Zip domain in HD-Zip subfamily I transcription
factors; a potential source of functional diversity
Agustín L Arce, Jesica Raineri, Matías Capella, Julieta V Cabello, Raquel L Chan
*
Abstract
Background: Plant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a
leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro
the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression
patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the
control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes.
Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing
information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition,
some of the results were also experimentally supp orted.
Results: A phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside
the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the
recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory
mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation.
A yeast one-hybrid experiment demonstrated that the activation activity of ATH B1, a member of one of the
groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member
with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the
chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein,
revealing the importance of this module inside the whole protein.
Conclusions: The bioinformatical results and the experiments conducted in yeast and transge nic plants strongly
suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their
function, hence potentially constituting a major source of functional diversity among members of this subfamily.
Background
Plant transcription factors
Transcription factors (TFs) play key roles in signal trans-
duction pathways in all living organisms. They are pro-

teins able to recognize and bind speci fic DNA sequences
(cis-acting elements) present in the regulatory regions of
their target genes. In general, these proteins have a mod-
ular structure and exhibit at least two types of domains: a
DNA binding domain and a protein-protein interaction
domain which mediates, directly or indirectly, the activa-
tion or repression of transcription [1].
In plants, s everal TF families have been identified but
only a relatively small number of members have been
functionally studied [2,3]. Such identification was per-
formed essentially in plants whose genome has been
sequenced, e.g. Arabidopsis, for which a comparison with
known ani mal TFs indicated the existence of about 2000
TFs [3,4]. TF families are classified according t o their
binding domain and divided in subfamilies according to
additional structural and functional characteristics [2,5-9].
The HD-Zip family of transcription factors
Among the identified TF f amilies, the HD-Zip family is
composed of proteins bearing a homeodomain asso-
ciated to a leuc ine zipper (hereafter, HD and HALZ),
* Correspondence:
Instituto de Agrobiotecnología del Litoral, Universidad Nacional del Litoral,
CONICET, CC 242 Ciudad Universitaria, 3000, Santa Fe, Argentina
Arce et al. BMC Plant Biology 2011, 11:42
/>© 2011 Arce et al; licensee BioMed Central Ltd. Thi s is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.o rg/licens es/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
association unique to plants. Due to this specific asso-
ciation and knowing that HD proteins in other king-
doms are involved in development, HD-Zip proteins

were proposed as key players in plant specific develop-
mental processes, such as those a ssocia ted to external
stimuli and stresses [10]. Four groups, named I to IV,
have been identified fundamentally based on four parti-
cular characteristics: sequence conservation within the
HD-Zip domain, the presence of additional conserved
domains, gene structure and the pathways in which
these proteins participate (for a review see [9] and [11]).
HD-Z ip III and IV members are, on average, the largest
proteins; they exhibit a START (STeroidogenic Acute
Regulatory protein-relatedlipidTransfer)andSAD
(START adjacent) domains towards the C-terminus in
relation to the HD-Zip domain [9], plus a MEKHLA
(called after the goddess of lightning, water and rain)
domain in subfamily III proteins [12]. HD-Zip II TFs
also have a distinguishing feature in their C-terminus,
the CPSCE motif responsible for redox regulation
of protein activity [13], and the ZIBEL motif in their
N-terminus [11]. No common feature outside the
HD-Zip domain has been assigned to subfamily I TFs.
What is known about HD-Zip subfamily I members
HD-Zip I group has 17 members in Arabidopsis thaliana
divided in six classes according to their phylogenetic rela-
tionships and intron/exon distribution: a (ATHB3, -20,
-13 and-23), b (ATHB1, -5, -6 and -16), g (ATHB7 and
-12), δ (ATHB21, -40 and -53), ε (ATHB22 and -51)and
 (ATHB52 and -54) [14]. The encoded proteins tested
for binding specificity in vitro recogni ze the same pseu-
dopalindromic sequence with the highest affinity [15-17].
This affinity, but not the specificity of this protein-DNA

interaction is affected by the aminoacids of the homeodo-
main N-terminal arm [18].
ATHB7 and AT HB12, coded by paralogous genes,
share 80% identity in the HD-Zip domain amino acid
sequence. Both genes are regulated by drought stress in
an abscicic acid (ABA)-dependent way [19,20]. Their
developmental expression pattern is similar but ATHB12
expression is detectable in lateral root primordia, young
leaves and inflorescence stems while ATHB7 is not, at
least under normal growth conditions. When ABA is
exogenously applied, their expression patterns overlap
[21,22]. The constitutive expression of ATHB7 in the
Wassilewskija (WS) genotype generates a developmental
delay and a characteristic morphological phenotype
(similar to the observed when WT plants are subjected to
drought) while the silencing of this gene apparently does
not alter the phenotype [21]. ATHB12 overexpressors are
similar to ATHB7 transgeni c plants [22]. Both transgenic
genotypes presented also increased lateral branching o f
the stem compared with the WT (WS) genotype. In both
cases the phenotype in roots is ABA dependent while the
phenotype in stems is ABA indepen dent [22,23]. The
characterization of athb12 mutants and ATHB12 overex-
pressing plants indicated that this gene product is some-
how inhibiting the expression of the gene encoding the
GA-20-oxidase, leading to the short stem phenotype due
to a reduction in gibberellins content [23].
Our research group has characterized HAHB4, a sun-
flower HD-Zip I protein sharing 60% and 53% identity
respectively with ATHB7 and -12 in the HD-Zip domain

[24]. However, HAHB4 has a short carboxy-terminal
region (CTR, 64 amino acids after t he HALZ) while
ATHB7 and 12 present 127 and 106 amino acids in this
region, respectively. HAHB4 expression is very low in nor-
mal growth conditions and it is up regulated in roots,
stems and leaves by ABA, mannitol, NaCl, drought and
darkness as well as by jasmonic acid (JA) and ethylene
(ET) [24-28]. The phenotype observed when this sun-
flower gene is ectopically expressed in Arabidopsis plants
strongly resembles that o f ATHB7/12 overexpressing
plants [29]. However, HAHB4 plants exhibited drought
tolerance and a senescence delay while ATHB7 and 12 did
not. Moreover, when HAHB4 seedlings were treated with
exogenous ACC (1-aminocyclopropane-1-carboxylic-acid,
a precursor of ET biosynthesis) the plants did not present
the typical triple response to et hylene [26]. This observa-
tion together with a microarray analysis indicated that
HAHB4 inhibits the expression of ethylene receptors and
thereafter the ability to sense this hormone [26,28].
Another pair of paralogous genes, ATHB13 and
ATHB23, code for proteins which share 78% identity in
the HD-Zip domain and 87 and 77% identity, respectively,
with
the HD-Zip domain of the sunflower HAHB1 [30].
The morphological characteristics of transgenic plants
expressing ATHB13 and HAHB1 genes under the CaMV
35S promoter are similar; e.g. serrated leaves, differential
cotyledons phenotype when grown in sucrose 4% [[31,32];
JV Cabello, AL Arce, and RL Chan, unpublished results].
Is the HD-Zip domain sufficient for the function of HD-Zip

I TFs?
The proteins encoded by the above mentioned genes
(i.e., ATHB12, 13, 23; HAHB4 and HAHB1), ATHB5,
ATHB1 and CPHB1 bind in vitro with maximal affinity
the same target sequence [15-17,33]. Notably when
transgenic plants in which these or other HD-Zip I
encoding genes were expressed in Arabidopsis under
the CaMV 35S promoter, the resultant phenotypes
were clearly different with the exception of those genes
phylogenetically closely related. These facts strongly
suggest that the function of these genes may be signifi-
cantly determined by other characteristics in addition
to differences in expression patterns and target gene
preferences.
Arce et al. BMC Plant Biology 2011, 11:42
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In this sense, previous works have supported the func-
tionality of the CTR s of HD-Zip I proteins. It was
shown that this portion of ATHB12 is capable of tran-
scriptional activation in ye ast one-hybrid experiments
[34] and functional complementation of a NaCl-sensitive
calci neurin (CaN)-deficient yeast mutant, only when the
protein has a complete CTR [35].
Sakuma et al. [36] identified HvHox2, a putative para-
logue of VRS1, by observing the effect caused in the
Hordeum vulgare spikelets development. These two
genes, both encoding HD-Zip I proteins, differ particu-
larly in the CTR. HvHox2 exhibits 14 additional amino
acids compared with VRS1. These authors identified a
conserved motif in this portion of the protein and sug-

gested that it could interact with certain classes of co-
activators in order to exert its biological function, as it
has been proposed for HAHB4 [36,37].
TL (Tendril-less) is a garden pea HD-Zip protein which
mutation (tl) generates plants with a particular phenotype:
tendrils are converted to leaflets, they are no longer inhib-
ited from completing laminar development. Notably, a
mutant in which this gene codes for a protein lacking 12
amino acids in its CTR exhibited the same phenotype as a
mutant unable to express the gene [38].
Based on the literature data and on our own observa-
tions we aimed to put in evidence that the CTRs and
NTRs (amino terminal regions) may be playing an impor-
tant role in the signalling networks in which HD-Zip pro-
teins participate, d etermining to some extent their
functionality. We used bioinformatics to detect new
sequence motifs in the NTRs and CTRs of the HD-Zip I
proteins. Further, we experimentally tested the function of
a CTR by making chimeric constructs and uncovered a
motif specific function.
Results
Phylogenetic analysis of HD-Zip proteins from different
species resolved six different clades
An in silico analysis was performed on a set of 178
sequences from HD-Zip I transcription factors from differ-
ent species (Additional file 1). They were selected merging
the database of proteins from species with sequenced gen-
omes [11] and a set retrieved from NCBI’sConserved
Domain Architecture Retrieval Tool (CDART).
The initial approach involved the construction of three

phylogenetic trees: the first with the subsequenc es com-
prising the HD and the HALZ domains of each protein
(named HZT), the second with this same subset plus
three HD-Zip II proteins from Arabidopsis which were
used as outgroup (HZT + OG), and the last with the
complete sequences of the proteins ( named CST). The
subset of sequences used for the HZT and HZT + OG
was obtained using HMMer [39] and the corresponding
HMM models [40].
The sequences were aligned with MAFFT (Additional
files 2 and 3) [41] and maximum-likelihood phylogenetic
trees constructed using PhyML [42] with 100 bootstrap
replicates for the HZT and HZT + OG, and 144 boot-
strap replicates for the CZT (Figure 1 and Additional
files 4 and 5). As expected, the three HD-Zip II proteins
formed a separate clade in the HZT + OG and its rela-
tive location was used to root the three trees.
The HZT was considered the reference tree because it
was constructed with a sequence alignment obtained
exclusively from the sites which are homologous to all
the HD-Zip I proteins analyzed. The initial strategy
involved the comparative analysis of the HZT and CST,
and the manual inspection of the alignment of the com-
plete sequences. Overall, major clades with moderate or
good statistical s upport in the HZT a nd CST had, with
some exceptions, a very similar composition. Sequence
alignments in the NTRs and CTRs revealed evident
sequence conservation for most proteins in each clade.
Based on both observations, a total of 137 proteins were
divided in six groups (I to VI, Figure 1 and Additional

file 4). As can be seen in Figure 2 and Additional file 6,
each group has a reasonably distinctive CTR with vari-
able-length stretches of highly conserved amino acids.
The informational content in these regions can be
appre ciated by the increase in bootstrap values for most
of these clades in the CST where the NTRs a nd CTRs
are considered (Table 1).
Grouping was mainly aimed at recognizing common
potentially functional characteristics in the sequences of
groups of HD-Zip I proteins. Consequently, although
group I had a high bootstrap support value, it was
further divided in three subgroups: Ia, Ib and Ic; accord-
ing to sequence con servation, particularly in the CTR
(Figure 2). Con versely, the conservation in the NTR and
CTR (F igure 2) of proteins from groups III and IV
together with the significant bootstrap values in the
CST supported grouping of clades of proteins with weak
bootstrap values in the HZT.
Groups I, II, III, V and VI were formed of proteins
from dicots and monocots, excluding the 27 proteins
from mosses, lycophytes, ferns and conifers; and 14 pro-
teins from dicots. The 17 TFs from the moss Physcomi-
trella patens formed a separate clade named Pp group.
The species with sequenced genomes h ad at least one
member in each group, with the exception of Poplar in
group III and Arabidopsis in group IV, the only group
exclusively formed of proteins from dicots.
The high conservation of key residues in the HD-Zip I
homeodomains suggests little target-sequence variation
Certain residues in the HD, particularly in the helix III and

a flexible N-terminal arm are important determinants of
the sequence preferentially bound by the HD-Zip I TFs
Arce et al. BMC Plant Biology 2011, 11:42
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Figure 1 Phylogenetic trees of HD-Zip I transcription factors. Maximum Likelihood phylogenetic trees were constructed using the amino
acid sequences of 178 HD-Zip subfamily I transcription factors from different plant species including monocots, dicots, mosses, ferns and
conifers. The HZT was constructed with the sequences of the HD and HALZ domains and is the reference tree. The CST was calculated with the
complete sequences. Clades highlighted with different colours represent groups of transcription factors sharing common motifs in their CTRs.
These clades are numbered from I to VI whereas group I is divided in three subgroups named Ia, Ib and Ic. Inside these groups, clades
exclusively formed by monocots or dicots transcription factors were labelled with an M or a D, respectively; and their structure was collapsed to

ease visualization. Proteins shared between groups in the HZT and CST have been erased from the CST. Unshared members have been marked
with an asterisk in the HZT. The group labelled Pp includes all the proteins from the moss Physcomitrella patens. Bootstrap support values, as
percentages, are indicated in the nodes. Branches with low bootstrap values (below 50%) have been collapsed, with the exception of the basal
branches of groups Ic, III and IV in the HZT which have further support from bootstrap values in the CST (see Table 1) and conserved motifs in
the NTRs and CTRs (Figures 2, 4 and Additional file 8).
Arce et al. BMC Plant Biology 2011, 11:42
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[18,43,44]. The alignment of the HD and HALZ sequences
corresponding to the proteins of the dataset analyzed
(Additionalfile2)showsaveryhighconservationofthe
amino acids in these homeodomain positions, i.e.: K2:
74%, K3: 94%, R5: 93%, I/V47: 54/46%, Q50: 100%, N51:
99% and R55: 100% (corresponding to the positions K4,
K5, R7, I/V57, Q60, N61 and R65 in the alignment, Addi-
tional file 2). This result suggests that target-sequence var-
iation may not be a major source of functional diversity
within the subfamily I of HD-Zip TFs.
HD-Zip proteins from each clade present conserved
motifs in their CTRs
Previous experimental evidence supporting the functional-
ity of the CTRs of a few HD-Zip I proteins [34-36,38] lead
us to further explore this region.Fromthealignmentof
the CTRs, the only evident feature was a bias in W com-
position towards the last residues of the protein. The his-
togram in Figure 3 shows that W was significantly
enriched in the final tenth part of the CTRs of the 178
proteins studied.
In order to deepen the analysis of the CTRs, a motif
discovery approach was conducted using the MEME
program [45]. A single run with all the sequences (with

a limit of 20 motifs and a minimum width of six sites)
yielded motifs with e-values ranging from 4.3e -279 to
3.9e-27. Figure 4 illustrates the motif composition and
location in each CTR; the sequence logos of each motif
are presented in Figure 5.
Most of the motifs found were highly or completely
group specific, only group VI lacked distinctive motifs.
Nonetheless, there is one clear exception: motif 2 appears
in most members of groups III, IV and V and in many
P. patens proteins. Its distinguishing features are: an
enrichment in Ser with two occupying conserved positions
separated by six residues, and several acidic amino acids.
On the basis of motif distr ibutio n, the CTR could be
roughly divid ed in two regions: a proximal regi on, adja-
cent to the HALZ; and a distal region, comprising the
final part of the protein. The former involved up to
three concatenated motifs adjacent to the HALZ (in
groups II, V, IV) and/or a motif located around the cen-
tral portion of the CTR (Figure 4); while the latter was
characterized by a motif covering the last residues,
which in groups Ic, II, IV, V and Pp was accompanied
by an adjacent motif towards the N-terminus (Figure 4).
The analysis o f the different motifs according to their
position and composition revealed a remarkable feature;
the presence of one or more Trp with high frequencies in
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Figure 2 Sequence logos of CTRs from the six groups identified. The sequence logos were constructed with the alignment of the CTRs of
the proteins belonging to each of the six groups, including subgroups Ia, Ib and Ic. The height of the residues correlates with their frequency in
the alignment, which allows the recognition of clearly conserved regions.
Table 1 Bootstrap values in the HZT and the CST
I Ia Ib Ic II III IV V VI
HZT 100 99 83 40 53 37 21 95 99
CST 90 100 69 93 69 82 63 pph 100
Bootstrap values for the different clades identified in the trees HZT and CZT.
pph paraphyletic.
Arce et al. BMC Plant Biology 2011, 11:42
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all the motifs at the end of the proteins (motifs 1, 3, 5, 9,
14, 19, Figures 2 and 5). Another aromatic amino acid
with high frequencies was Phe, present in most of the
motifs in the distal region (motifs 1, 3, 5, 7, 10, 14 and 20).
Additionally, many positions were occupied by acid resi-
duesandafewbyPro(motifs1,3,5,7and9).This
sequence features highly resemble those of AHA motifs
found in HSF (
Heat Stress Transcription Factors) TFs [46].
In the motifs found in the proxima l region of the CTR,
the residues with the highest frequencies were Ser and
acidic amino acids (Figure 5). Since Ser are potential
phosphorylation sites and transcription factors constitute
preferential can didates for this type of modification [47],
we explored the predicted possibility of phosphorylation
inSer,ThrandTyrwiththeprogramNetPhos2.0[48].

Using a cutoff score of 0.9, the results showed that many
of the high-frequency Ser in these motifs are predicted
targets of phosphorylation, particularly those present in
motifs 2, 4, 6, 7, 10, 12, 16, 17 and 18 (Figure 5 and Addi-
tional file 7), most of which were in the proximal region
of the CTR (Figure 4).
Interesting results were obtained when another type of
putative post-translational modification was analyzed,
sumoylation. SUMO is mainly conjugated to the K in
the motif ΨKXE/D (Ψ , large hydrophobic residue; X,
any amino acid; E/D, Glu or Asp) [49]. This peptide
appears with a high frequency in motifs 6, 8, 10 and 12;
the last present in the proximal region and the other in
the distal region, adjacent to the terminal motif. To
further address this observation, we searched for the
degenerated motif in all the CTRs, Ψ being F, V, I, M or
L. The motif was found 143 times in 95 of the 178 pro-
teins. Moreover, the last position was mostly E: the
motif ΨKXEcorrespondsto120ofthe143motifs
found, and they are distributed in 92 of the 95 protei ns.
There was also a bias towards the identity of the hydro-
phobic residue: V > I > L > M > F (62% > 19% > 11% >
6% > 2%). The sumoylation motifs were mainly present
in groups I (b and c), II, V and the Pp group (Figure 4).
In groups II and V they were found twice per protein.
As a rudimentary test of the significance of these
results, the motif ΨKX-[ED] in which the last position
could be any of the 20 amino acids but Glu or Asp was
searched.Atotalof82motifsin63proteinswere
found, which compared to the appearances of the cano-

nical motif (143 motifs in 95 proteins, (ΨKXE/D)/(ΨKX-
[ED]): 1.74) puts in evidence the overrepresentation of
putative SUMO conjugation sites.
The NTRs also present some conserved motifs
The NTRs were analyzed applying a similar motif-dis-
covery strategy. The program MEME elicited 12 motifs
with e-values ranging from 2.6e-231 to 3.7e-4. Motifs
logos and distribution are illustrated in Additional files
8 and 9. Group definition was somehow supported by
this distribution, with some exceptions. Motif 1 is widely
distributed appearing in groups II (dicots only), III, IV
and Pp. Subgroups Ia, Ib and Ic lacked distinctive
motifs, and group II was divided in monocots and dicots
by unshared motifs. It should be noted that group VI,
which had no distinctive motifs in the CTR , was distin-
guished by motif 10 in the NTR.
In the attempt of finding putative functional signifi-
cance to the motifs of the NTR, the program NetPhos
was employed to predict probable phosphorylation sites
with a c utoff of 0.9 (Addition al files 8 and 10). The Ser
residues in motifs 1 (mostly from group I), 3 and 6 (posi-
tion 10 with high frequency) are the best candidates for
this post-traslational modification because they are also
highly conserved.
The program NLStradamus [50] was used to predict
nuclear localization signals (NLS) in the complete pro-
teins. This signal was found only in 16 of the 178 pro-
teins; among them, three had it in the CTR (ATHB54,
Pp_sca_35 and P p_sca_143, all three abnormally long
HD-Zip I proteins), and the other 13 in the NTR. Of

these 13, six NLSs belonged to proteins from group VI
(11 members) and fell within motif 10, found in 10 of
the members (Additional files 8 and 11).
In order to make a comparison with the sumoylation
results obtained with the CTRs, the motif ΨKXE/D was
searched in the NTRs. Only eight motifs were found
(Additional file 8), seven exhibit a Glu in the last position
and four of them a Val in the first position. Despite amino
acid frequencies showed some analogy with those in
Figure 3 Frequencies of tryptophans in the CTRs. The histogram
represents the frequencies of Trp within the CTRs of the 178
proteins according to their relative position in this region, which
was divided in ten parts. The last tenth shows a visible enrichment.
Arce et al. BMC Plant Biology 2011, 11:42
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sumoylation motifs found in the CTRs; the number of
sites found is negligible to consider sumoylation an impor-
tant general modification in HD-Zip I NTRs. To reinforce
this conclusion, the motif ΨKX-[ED] was searched in the
NTRs: it appeared 59 times in 53 proteins ((ΨKXE/D)/
(ΨKX-[ED]): 0.14), in contrast with the results obtained
with the CTRs ((ΨKXE/D)/(ΨKX-[ED]): 1.74).
ATHB1 CTR acts as an activation domain in yeast cells
In order to determine the putative activator action of the
CTR mot if in these TFs, one member of group III,
ATHB1, was analyzed. Genetic constructs in which the
whole cDNA or a mutated version, where the CTR was
deleted, were obtained and yeast cells (AH109) were
transformed with these as well as with the appropriate
control constructs (Figure 6A). The positive colonies

grown in the medium lacking Trp were transferred to a
medium lacking His in which only the cells with the abil-
ity to t ransactivate can grow. Cells bearing the complete
cDNA or just the CTR grew in this medium while the
cell s transf ormed with the truncated const ruct and those
transformed with the empty vector did not (Figure 6C).
Figure 4 Motif location in the CTRs. The 20 motifs found by the program MEME are depicted according to their locatio n in each CTR. The
identity of each motif is colour coded according to the legend. Groups are highlighted with a box of dashed boundaries and the phylogenetic
relations between the proteins are indicated by the tree on the left side of the plots. Putative phosphorylation sites (Ser, Thr, Tyr) are marked
with a black diamond and sumoylation motifs with a blue inverted triangle.
Arce et al. BMC Plant Biology 2011, 11:42
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The empty vector bears the ADH1 promoter directing
the expression of the GAL4 transcription factor DNA-
binding domain; this construct is not able to transactivate
and therefore, the cells transformed with it cannot live in
a selective medium. Colonies w ere also tested for b-
galactosidase activity and the results supported the
growth assay (Figure 6B). These observations indicated
that the CTR is the region responsible for the transactiva-
tion activity of this TF, at least in yeast.
The phenotype of the plants transformed with chimerical
constructs is similar to that of the plants transformed
with the CTR donor protein
In order to determine the importance of the CTR in the
structure/function relationship of HD-Zip proteins, we
have chosen two well characterized members of this tran-
scription factors family to perform chimerical constructs
and evaluate the phenotypes in transgenic plants.
HAHB4 inhibits the triple response to ethylene when it is

ectopically expressed in Arabidopsis while HAHB1, like
its h omologue ATHB13, confers a serrated shape to
leaves [[26], JV Cabello, AL Arce; and RL Chan, unpub-
lished results]. In relation to the in sili co analysis,
HAHB1 fell in group V and HAHB4 in group I, outside
the three subgroups with c haracteristic CTRs. No motifs
were found in HAHB4 NTR (relatively small, 19 amino
acids) or CTR (62 amino acids); it has two Trp in the
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Proximal region
Distal region
Adjacent to C terminal motif
C terminal portion
Adjacent to HALZ
Central portion

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Figure 5 Sequence logos of the motifs found in the CTRs. The sequence logos of the 20 motifs found in the CTRs are sorted according to
their relative position. To reflect chemical properties in the distal region, the motifs present in the same row are also combined in many CTRs
(except for motifs 9, 10, 19 and 20; some alternative combinations to those shown also exist).
Figure 6 ATHB1 CTR acts as an activation domain in yeast
cells. (A) The complete sequence of ATHB1, a version without the
CTR (ATHB1WCT), and the CTR alone (ATH1CT) were fused to the
DNA-binding domain of GAL4 (GAL4-BD). The empty vector
expressing only the GAL4-BD was used as negative control. (B) A b-
galactosidase activity assay. (C) Confirming this results, only the CTR
and the complete ATHB1 protein had the transactivation activity
required to reverse the auxotrophy to His of the AH109 yeast cells,

allowing them to grow in medium lacking this amino acid.
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 8 of 19
final residues, with the particularity of being adjacent to a
Lys, not usual in AHA motifs. HAHB1 possesses motifs
13, 2, 11, 20 and 1 in the CTR (122 amino acids); and
motifs 2, 3, 6 and 4 in the NTR (91 amino acids).
Mutant and chimerical genetic constructs were per-
formed to evaluate the CTR functionality. The CTR of
HAHB1 was fused to the HD-Zip of HAHB4 (protein
H4-H1)andbothcDNAsweredeletedintheirCTRs
forming H1WCT (HAHB1 without CTR) and H4WCT
(HAHB4 without CTR), as dep icted in Figure 7A. Fused
to the 35S CaMV promoter, these constructs were used
to transform Arabidopsis plants. Three independent
lines of each genotype presenting differe ntial expression
levels were chosen for further analysis (Figure 7B).
Seedlings were grown in 5 μM ACC, an ethylene precur-
sor, and photographs taken when they were four-day-old.
Figure 7C illustrates the phenotype observed for sensitive
and insensitive plants while in Figure 7D the proportions
of insensitive plants in eight groups of 20 plants from each
line subjected to this treatment is depicted with a box
plot. Trans genic plants with high expressio n levels of
HAHB4 (lines B and C) were used as controls and did not
show the apical hook, as expected, while a low expression-
level line (line A) presented a high percentage of ACC sen-
sitive plants. H4WCT exhibited a moderate insensitivity to
ACC. H1WCT and H4-H1 plants showed more sensitivity
than H4WCT plants. Finally, the plants which displayed

the higher sensitivity to ACC treatment were HAHB1 and
WT, showing a very low percentage of seedlings without
apical hook (Figure 7D).
Notably, H4-H1 plants were more sensitive to the
ACC treatment than HAHB4 plants but not as sensitive
as HAHB1 plants, while H1WCT plants decreased their
sensitivity to the treatment. Together these observations
indicate that the CTR of HAHB1 in H4-H1 seriously
impai rs the physiol ogical response triggered by HAHB4,
more effectively than the removal of its own CTR (i.e.,
in H4WCT plants). In fact, H1WCT could, to some
extent, mimic the physiological response of HAHB4
plants to ACC, questioning the degree of participation
of the CTR when HAHB4 is involved in this pathway.
The phenotype of rosette-leaf serration was also
tested. The number of serrations per leaf was calculated
for high expression lines of each genotype: WT, HAHB1
B, HAHB4 B and H4-H1 A, B and C plants. The results
showed that HAHB1 B and H4-H1 B plants presented a
clear increase in serration while the rest of the lines had
a serration similar to t hat of WT plants (Figure 8). The
quantifications were subjected to the Kruskal-Wallis
one-wayanalysisofvariancebyranksandthenthedif-
ferent lines were classified in groups according to pair-
wise comparisons with a p-value of 0,05 (Table 2). The
results indicated that HAHB1 and H4-H1 B had a statis-
tically significant increase in serration. Together with
H1-H4 A, these three lines were distinguishable from
HAHB4 plants.
Discussion

Transcripti on factors are modular proteins par excellence
[51]. Among the many types of modules present in differ-
ent TFs, two are almost indispensable: a DNA-binding
domain and a protein-proteininteractiondomainwhich
mediates activation or repression of transcription [1].
HD-Zip proteins are transcription factors unique to
plants and since the isolation of the first member in 1992
[9,52], several works have informedthattheprotein-
DNA interaction m ediated by the HD is highly specific
and needs as a prerequisite the dimerization of the TF
through the HALZ [16,17,53]. Other domains outside the
HD and HALZ are present in members from HD-Zip
subfamilies III and IV (e. g., START, SAD domains; [9]).
HD-Zip II TFs have a redox motif in their CTRs [13] and
a Ziebel motif in their NTRs [11]. In the case of HD-Zip
I proteins, no additional domains or motifs have been
described for the whole group. Some reports suggested
thepresenceofanuclearlocalization sequence in their
amino terminus [54]; however, no definite experimental
evidence in this sense has been presented thus far. A few
reports have provided evidence ind icating a fu nction for
the CTR of these proteins. In this sense, activation activ-
ity was demonstrated as dependent on the CTR of
ATHB12 in yeast [34]. A dditional support to the impor-
tance of the CTR was provided by Sakuma et al. [ 36];
they identified that the recessive allele vrs1, which causes
the six-rowed phenotype in barley, encodes an HD-Zip I
TF 14 residues shorter in the CTR than its paralogous
gene HvHox2 (both share 88% of identity in the whole
protein), which was caused by a 300-bp insertion that

introduced a stop codon. These authors ident ified a con-
served motif within these 14 amino acids and suggested
that this motif could interact with certain classes of co-
activators in order to exert its biological function [36,37].
Recently, a pea deletion mutant in one HD-Zip pro-
tein, in which tendrils were converted into leaflets (they
were no longer inhibi ted from completing laminar
development), was shown to exhibit the same phenotype
as a mutant in which the 12 ami no acids of its CTR
were not translated [38].
The starting point of our analysis was a 178 HD-Zip I
protein dataset retrieved from CDART NCBI’s database
and that generated by Mukherjee et al. [11]. The first step
involved the construction of three phylogenetic trees: the
HZT with the HD and HALZ domains, the HZT + OG in
which three HD-Zip II proteins were added as outgroup,
and the CST with the complete sequences. The HD-Zip II
TFs formed a clade which relative position was used to
root the 3 threes. The HZT was considered the reference
tree as its construction only used the sites homologous to
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 9 of 19


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





 

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

A BC ABC ABC ABC ABC WT
0.0
0.2
0.4
0.6
0.8
1.0
Ethylene−treated seedlings
Pl

ants w
i
t
h
out
h
oo
k

[
proport
i
on
]
Lines
HAHB4
H4WCT
H4−H1
H1WCT
HAHB1

Figure 7 Triple response to ethylene in chimerical transgenic plants. (A) Schematic representa tion of the different constructs used to
transform Arabidopsis plants (B) Relative expression levels of the different transgenes in independent lines measured by qPCR. The line with the
lowest expression was assigned an unitary level (1). (C) The sensitivity to ethylene was measured analyzing whether the seedlings developed
apical hook (sensitive) or not (insensitive). The image exemplifies the phenotypes observed. (D) The results for three different lines from each
genotype are presented in the boxplot.
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 10 of 19
all proteins. With the objective of identifying relevant
functional regions outside the HD and HALZ domains,

the sequence alignments of the NTRs and CTRs were
inspected and the clades formed in the HZT and CST
were compared. Based on these observations, six groups
were identified (I to VI, Figure 1). The evident similarity in
their CTRs, shared by most members of each group
(Figure 2 and Additional file 6), strongly suggests the com-
mon ancestry of these proteins. This observation was sta-
tistically weakly supported for groups Ic, II, III and IV in
the HZT; but was significantly supported by bootstrap
values in the CST (Table 1). This suggests that t he
sequence of the HD and HALZ may not be sufficiently
informative to clearly resolve some clades, thus the con-
struction of a tree with the complete sequences proved to
be a valuable strategy t o identify and provide additional
support to the hypothesis of relationship among t he
proteins in the different groups. Nonetheless, as the CST
was constru cted considering sites which are homologous
only to subgroups of proteins, the phylogenetic recon-
struction lays over a partially unfulfilled hypothesis of
homology and the tree was not taken as the reference
reconstruction. None of the six groups identified included
proteins from species of non-flowering plants; they
included only proteins from monocots and dicots (except
for group IV exclusively formed of proteins from dicot s).
This suggests that a common ancestor of the genes encod-
ing these proteins existed prior to the split of monocots
and dicots, but no clear homologues could be found in the
analyzed proteins from more basal species.
The 17 pr otein s from Physcomitrella patens formed a
separate clade. This is potentially due to the low diver si-

fication of subfamily I HD-Zip TFs in this non-vascular
plant. Rooting positioned this g roup as a non-basal
group within c lades of mostly flowering plant proteins.
If the hypothesis of multiple gene losses in this species
is considered unlikely, this presents incongruence with
the known species phylogeny. This observation together
with the relatively poor statistical support in the basal
part of the tree (beyond the six groups identified) sug-
gests that the precis e relationship among groups could
not be fully resolved. More sequences or a more com-
plex approach might be needed to achieve it.
In the phylogeny of Arabidopsis HD-Zip I proteins
obtained by Henriksson et al. [14] a classification in six
classes was made. Proteins from class a (ATHB3, -20,
-13 and-23) were included in group V, three proteins
from class b (ATHB5, -6 and -16) were included in
group I I while ATHB1 fell in group III, proteins from
class g (ATHB7 and -12) were contained by group Ic,
and proteins from class δ (ATHB21, -40 and -53) were
included in group VI. None of the six groups included
proteins from classes ε (ATHB22 and -51) and 
(ATHB52 and -54), moreover, the monophyly of class 
in relation to other Arabidopsis proteins is not pre-
served in the HZT (i.e., when phylogenetic relationship s
among Arabidopsis TFs is reconstructed from this tree,
this class does not constitute a separate clade). Although
the reconstruction of Henriksson et al. [14] is supported
by additiona l informa tion, (e.g., intron/exon distribution
within the HD a nd HALZ domains), it should be noted
that classes ε and  are the only for which no support

from duplication history was found by the authors. It
should also be mentioned that ATHB54 is a very atypi-
cal HD-Zip I protein with an extremely long CTR (325
amino acids) in which a RNA recognition motif can be
identified.
As mentioned before, HD-Zip I proteins tested in
vitro bind specifically and with high affinity the same
pseudopalindromic sequence CAAT(A/T)ATTG [16,17].
The alignment of the HD and HALZ of the proteins in
Figure 8 Leaf s erration phenotype. Boxplot illustrating the
serration grade of the leaves belonging to transgenic plants
transformed with different constructs. The images are examples of
the analysis conducted with the leaves (HAHB1 on the right and
HAHB4 on the left).
Table 2 Pairwise comparisons after Kruskal-Wallis test
Transgenic line Rank Groups
HAHB4 B 50.36 A
H4-H1 C 58.53 A B
WT 66.88 A B
H4-H1 A 78.53 B
H4-H1 B 120.03 C
HAHB1 B 143.17 C
The sum of ranks of the Kruskal-Wallis test for each line is presented together
with the groups resulting from the pair wise comparison. Lines with
statistically different behaviours do not share the same group.
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 11 of 19
the studied dataset showed a high degree of conserva-
tion for the residues responsible for sequence binding
specificity [9]. Consequently, the putative abi lity of tar-

geting and regulating different groups of genes may be
significantly determined by other protein features out-
side the HD.
Numerous studies demonstrated that the expression of
HD-Zip I proteins under the control of the CaMV 35S
promoter [21-23,26-29,55] results in plants with diverse
phenotypes. But considering that their DNA-binding
specificity could be a minor source o f functional varia-
bility, conclusion supported by our in silico analysis of
the 178 protein dataset; the importance o f the role of
the poorly characterized NTRs and CTRs in the func-
tion of HD-Zip I TFs could have been overlooked. The
sequence conservation found in these do mains and the
identification of different groups of TFs are in agree-
ment with this notion.
In order to gain insight into the function of the con-
served regions, a motif discovery strategy was carried
out. As a result of running the program MEME on the
NTRs and CTR s, 12 and 20 moti fs were obtained,
respectively. Many motifs were group specific/cha racter-
istic (Figure 4 and Additional file 8) but a few were
more widely distributed. In general, the CTRs displayed
more correlation betwee n groups and motifs, although
in this analysis no specific motifs were found in the
CTR for group VI and the only motif present in sub-
group Ib (monocots) was also in subgroup Ic (dicots).
The motifs in the NTRs were only specific for groups II
(being in monocots different than in dicots), VI
(opposed to what happened in the CTR), V and Pp.
GroupsIIIandIVsharedmotifsintheNTRandsub-

groups Ia, Ib and Ic had no characteristic motifs in this
region.
Based on motif distribution within each CTR, the
domain was divided in two regions: proximal and distal .
In the distal region, motifs had up to three Trp occupy-
ing p ositions with high frequency (mo tifs 1, 3, 5, 9, 14,
19), particularly those corresponding to the last residues
of the protein. In some motifs the same occurred with
Phe (motifs 1, 3, 5, 7, 10, 14 and 20) and Pro (motifs 1,
3, 5, 7 and 9). Another characteristic of the motifs in
the distal region was the high abundance of acidic
amino acids. These characteristics strongly resemble
those of AHA activator motifs (
Aromatic, l arge Hydro-
phobic,
Acidic context) of the type found in HSFs pro-
teins [46,56] and other transcriptional activators [57].
Mutational analysis in AHA motifs from HSFs has pro-
ven that importance of aromatic and l arge hydrophobic
residues in the core positions of this motif, which for
these proteins were generally Trp and Phe. The role of
AHA motifs in the interaction with proteins from the
basal transcription machinery (i.e., SWI/SNF, TFIID and
SAGA complexes) has also been demonstrated [46,56].
The presence of these motifs in most HD-Zip I proteins
constitutes an important finding for dif ferent reasons.
Firstly, it provides solid evidence that most TFs from
this subfamily act as transcriptional activators, as has
been demonstrated experimentally for s ome proteins
previously [14,34,58]. Secondly, it allows the location of

the specific region within the CTR, the distal region,
which is acting as an activation domain. Good examples
of the importance of this regions are VRS1 [36], which
lacks a motif in the CTR in relation to HvHox2, and the
protein without the last 14 residues of the CTR that
mimics the tl mutation [38]; in both cases the deletions
corresp ond to AHA-like sequences. Finally, the different
versions of AHA motifs found, shared by the members
of each group, may be responsible for the interaction
with different co-activators or members of the basal
transcriptional machinery [37], and thus provide a
source of functional divergence among HD-Zip I pro-
teins together with differential expression patterns in
some cases.
In the proximal region of the CTR, many motifs were
characterized by the presence of Ser and acidic amino
acids occupying most of the high-frequency positions.
Many of the Ser in these motifs (i.e., motifs 2, 4, 6, 7,
10, 12, 16, 17 and 18) were predicted as putative phos-
phorylation sites. Particularly interesting wa s the case of
the widespread motif 2 in proteins from groups III, IV,
VandPp, always in the proximal reg ion. This may be
important as it has been previously demonstrated in
vitro [58] that the phosphorylation of ATHB6 with the
PKA kinase inhibits its DNA-binding activity.
Putative sumoylation sites were also investigated in the
CTR. The peptide to which SUMO is conjugated, ΨKXE/
D, was mainly present in motifs 6, 8, 10 and 12 which
were identified in groups Ic, II, V and Pp.Amore
exhaustive exploration revealed that 95 of the 178 CTRs

had this motif, which appeared 143 times. The acidic
amino acid was in most cases Glu (84%) and the hidro-
phobic residue, Val (62%).
The NTR presented 12 motifs and the correlation with
putative phosphorylation sites was also analyzed. Some
potential residues for this post-translational modification
were found in motifs 1 (mostly from group III), 3 and 6.
In relation to the motif 10 specific to group VI, six of
its 11 members were predicted t o have a NLS within
this motif characterized by a patch of basic residues.
This result may be extended to the other members of
the group as they share the same mot if. Sumoylation
was also analyzed in the NTR but only eight sites were
found, too few to consider it a significant general modi-
fication in HD-Zip I NTRs.
Proteins in the Pp groupsharedmotifsintheNTR
and CTR which supports the hypothesis that there has
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 12 of 19
been little diversificationofHD-ZipITFsinthisspe-
cies. This is probably related to the simpler tissue orga-
nisation of mosses. There may be also a high tendency
to heterodimerize and high functional redundancy
among members. Further analysis would be necessary to
address these observations.
The functionality of the CTR was also tested experi-
mentally. ATHB1 was selected as a characteristic mem-
ber of group III showing a typical AHA motif. It was
previously demonstrated that th is TF presents transacti-
vation activity in plant protoplasts [59]. A yeast one-

hybrid experiment was performed with the whole pro-
tein, the CTR alone, and a truncated version in which
the CTR was remove d. As predicted, t he removal of the
CTR containing the AHA motif generated a mutant
protein which lost the ability to transactivate in this sys-
tem, while the CTR alone was capable of transactivating
when fused to GAL4-BD (Figure 6).
In our laboratory, two sunflower HD-Zip I TFs have
been extensively studied, HAHB1 and HAHB4 [[26-29],
JV Cabello, AL Arce , and RL Chan, unpublished results].
Both bind in vitro the pseudopalindromic sequence
CAAT(A/T)ATTG, and both have been expressed in
Arabidopsis under the control of the 35S CaMV promo-
ter. The resulting plants exhibited clearly different phe-
notypes. Phylogenetically, HAHB1 is a member of group
V whereas HAHB4 is in group I, but not included in any
subgroup. HAHB1 NTR and CTR have most of the
motifs present in other members of its group, but no par-
ticular motifs were detected in HAHB4 NTR or CTR,
nevertheless, it has two Trp at the C-terminus, but with
an abnormal basic residue close to them. In addition to
the constructs of HAHB1 and HAHB4 complete coding
sequences, three constructs were prepared and Arabidop-
sis transgenic plants t ransformed: a version of HAHB1
without the CTR (H1WCT ), an analogous protein where
the CTR of HAHB4 was deleted (H4WCT), and a chime-
rical protein bearing the NTR, HD and HALZ of HAHB4
fused to the CTR of HAHB1 (H4-H1). The promoter was
also the 35S CaMV. The phenotypic differences between
the plants were assessed in a triple response experiment

in which hook formation in plants g rown in ACC was
measured as an indicator of ethylene sensitivity. The
results confirmed previous observations: HAHB4 plants
were highly insensitive and HAHB1 plants had a normal
response. Among the mutant proteins, H4WCT dis-
played the higher insensitivity while H1WCT and H4-H1
showed an intermediate response. The deletion of
HAHB1 CTR in H1WCT was enough to confer an inter-
mediate insensibi lity to the transgenic plants, in contrast
with the responsiveness displayed by HAHB1 plants. In
the case of H4WCT, the insensitivity is not completely
reverted by the deletion of the CTR. H4-H1 plants
showed some impairment in ethylene response, which
puts in evidence that the exchange i n CTRs has an
impact on the phenotype, at least for HAHB4 CTR which
has no motifs i n common. Despite these observations,
the fact that both mutant proteins lack ing the CTR (i.e.,
H1WCT and H4WCT) exhibit an altered sensitivity to
ethylene may suggest that HAHB4 CTR could be non-
essential for its activity in this pathway. Moreover, the
sole removal of the CTR of HAHB1 produces a protein
that does not develop the same response as HAHB1 in
transgenic plants, stressing the importance of the CTR in
this protein.
When leaf serration was evaluated it was shown that
the fusion of HAHB1 CTR with HAHB4 HD-Zip was
capable of generating the increase serration phenotype
in one of the transgenic lines, as was observed for
HAHB1 plants. The results obtained with the const ruc-
tion H4-H1 were not conclusive; a possibility is that

both domains, the NTR and the CTR, may be important
to generate a protein functionally similar to HAHB1.
Wenkel et al. [60] demo nstrated that small leucine zip-
per-containing proteins were responsible for the inhibi-
tion of HD-Zip III proteins by heterodimerization. It is
tempting to hypothesise that the capability of H1WCT of
mimicking HAHB4 and H4WCT insensitivity to ethylene
is the product of an inhibitory mechanism important in
this pathway, especially considering that HAHB4 has an
atypical AHA motif with a basic amino acid. In this sce-
nario, the native HAHB4 protein would be more efficient
than the mutant proteins H4WCT and H1WCT in exert-
ing this inhibitory activity.
Conclusions
The analyses of a set of 178 HD-Zip I proteins allowed
the identification of six groups, in most cases with high
sequence conservation outside the HD and HALZ. An
exhaustive exploration of these regions revealed an
AHA motif in the CTR of most proteins that could be
performing the activation role at a molecular level, like
in HSFs TFs; and possibly giving some specificity to the
interactions with the basal transcription machinery.
Putative phosphorylation sites were found in the NTRs
and CTRs, potential sumoylation motifs were discovered
in the CTR, and NLSs found in the NTR for the mem-
bers of one group. Altogether, this data allows us to
postulate an enriched model of HD-Zip I functional
domains or regions (Figure 9).
The presence of shared motifs in nearly all HD-Zip I
proteins of the moss Physcomitrella patens, a much sim-

ple organism than higher plants, points to an ancestral
low functional diversification of these proteins, beyond
the numerous genes present in its genome. In more
complex plants, like monocots and dicots, a discrete and
probably incomplete number of groups has been identi-
fied. At a functional and evolutionary level, the potential
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 13 of 19
significance of this division in groups represents an
interesting topic for further research.
Methods
Database searches and sequence retrieval
The amino acid sequences of subfamily I HD-Zip mem-
bers from different plant species were retrieved from
NCBI’s Conserved Domain Architecture Retrieval Tool
(CDART, [61]. Members whose genome has been
sequenced, i.e. Arabidopsis thaliana, Populus tricho-
carpa, Oryza sativa, Zea mays, Physcomitrella patens
and Selaginella moellendorffii were removed and
replaced with the sequences from these species obtained
by Mukherjee et al. [11]. An examination of these
sequences revealed that the tripeptide “ YES” had been
accidentally erased from the sequences published in this
work. This was confirmed by the authors (Bürglin, TR;
personal communication) and the sequences were cor-
rected. Sequence redundance was checked using the
“skipredundant” program of the EMBOSS package [62]
and the results were manually inspected and curated.
Sequences with truncated C-t ermini not belonging to
the species mentioned were removed. Finally, three

sequences from sunflower and Medicago truncatula (i.e.,
MTHB1, HAHB1 and HAHB11) were manually added.
The final dataset is in Additional file 1.
Data alignments and phylogenetic analysis
The 178 sequen ces were aligned using MAFFT v6 with
the L-INS-i algorithm [41]. The result was manually
inspected and edited using the Seaview4 multiple
sequence editor [63]. Prior to phylogenetic analyses, the
ProtTest program [64] was used to select the best-fit
model of protein evolution, resulting in the JTT + I + G
model [65-67]. Phylogenetic reconstruction was per-
formed with this model plus the maximum-likelihood
algorithm using the PhyML 3.0 program [42] with the
NNI tree topology search operation and a total of 144
bootstrapped datasets. The phylogenetic tree shown in
Figure 1 and Additional files 2 and 3 was obtained with
the “ consense” program from the PHYLIP package [68]
and t he images generated with FigTree (.
ed.ac.uk/software/figtree/).
The HD-Zip domain, NTRs and CTRs of t he proteins
were obtained recognizing first the HD and LZ domains
with HMMer v2.3.2 [39] using the corresponding PFAM
HMM models (PF00046.21 and PF02183.10, [40]). Then,
the reports were parsed and the sequences processed
with the aid of Perl scripts in which different modules
from the Bioperl toolkit [69] were used. In this way
three datasets were constructed, one for each portion of
the protein : NTRs, HD-Zip domains and CTRs. With
thesequencesoftheHD-Zipdomainsaphylogenetic
reconstruction was performed as previously described

for the tree with the complete sequences; the boot-
strapped datasets were 100 (Additional file 2). Analo-
gously, a third tree was constructed including in the
alignment the HD-Zip domain of 3 HD-Zip II proteins:
HAT1, HAT22 and ATHB17; as outgoup (Additional
file 3). Their relative position in the tree was used to
root the three trees mentioned.
The logos shown in F igure 2 were produced with
WebLogo [70].
Motif discovery and functional predictions
The program MEME [45] was employed on the NTR
and CTR datasets. Some parameters were manually
adjusted as follows: the motif limit to 20, the minimum
motif width to 6, the minimum number of sit es for each
motif to 6, and the e-value threshold to 0.1. The plots
of motif distribution ( Figure 4 and Additional file 8)
were prepared with the genoPlotR package [71] for the
R statistical language [72].
The p rediction of potential phosphorylation sites was
performed on the NTRs and CTRs sequences of the 178
HD-Zip I proteins. For this purpose the stand-alone ver-
sion of NetPhos 3.1 [48] was used. The selected cutoff
score was 0.9. To better visualize of the results, in Addi-
tional files 7 and 10 each protein is represented by three
stacked sequences: i) only the residues predicted to be
phosphorylated are visible, the remaining are substituted
with dots; ii) the visible residues correspond to motifs
found by the MEME program; and iii) the complete
sequence is visible. For the prediction of NLSs the pro-
gram NLStradamus [50] was employed on the complete

sequences of the 178 proteins with a subsequent thresh-
old of 0.6. The Additional file 11 is the output report.
SUMO
NTD
HD-Zip
CTD AHA
DNA binding
Dimerization
Regulatory
(sumoylation,
phosphorylation)
Regulatory
(phosphorylation,
NLS)
Activation
PP
RNA
pol II
L L
L L
L L
L L
L L
Figure 9 Proposed model of functional domains in HD-Zip I
transcription factors. The in silico analysis conducted on a
considerably large dataset of HD-Zip I transcription factors allowed us
to postulate a generalized functional model of this family of proteins.
This is supported to some extent by previous studies and the
experimental results presented in this work. The well characterized
HD-Zip domains are in charge of DNA binding and dimerization, an

AHA motif in the CTR is responsible for activation, and the NTR and
CTR are regions potentially phosphorylated and sumoylated,
depending on the group, thus playing a regulatory role.
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 14 of 19
Constructs
35SCaMV:HAHB4 construct was previously performed
[29].
35SCaMV:HAHB1:TheHAHB1 cDNA was isolated
from a library constructed in lambda gt10 as previously
described [30]. This fragment was cloned in the EcoRI
site of the pMTL22 vector and restricted with BamHI/
SacI in order to clone it in the pBI 121 plasmid pre-
viously treated with the same enzymes. In this way,
HAHB1 expression is controlled by the 35S CaMV pro-
moter (JV Cabello, AL A rce, and RL Chan, unpublished
results).
35SCaMV:H4WCT: the HAHB4 cDNA without its
CTR was obtained by PCR amplification on the
35SCaMV:HAHB4 clone with oligonucleotides Transfl
and H4-WCT-R (see Table 3) and directly cloned in
pBI121 by previously restricted with BamHI/SacI.
35SCaMV:H1WCT:theHAHB1 cDNA without its
CTR was obtained by PCR amplification on the
35SCaMV:HAHB1 clone with oligonucleotides H1-
WCT-R and H1atF (see Table 3) and directly cloned in
pBI121 previously restricted with BamHI/SacI.
35SCaMV:H4-H1: to ob tain the chimerical construct,
two separate amplifications were performed generating
overlapping DNA segments as described by Higuchi et al.

[73]. The 5’ region of HAHB4 comprising the HD-Zip
encoding domain was amplified with oligonucleotides
Transfl and H1*H4R from the plasmid containing 35S:
HAHB4 in the pBI vector and the HAHB1 3’ region
encoding the carboxy terminus was amplified using
35SCaMV:HAHB1 in pBI121 and the oligonucleotides
H1*H4F and H1CDS-R (Table 3). Both products were
electrophoretically purified and after a cycle of denatura-
tion and hybridization, the hybrid was extended by Kle-
now and amplified by PCR using oligonucleotides Transfl
and H1CDS-R. The final PCR product was purified from
agarose and cloned into the pCR R2.1-TOPO vector
(Invitrogen). Finally, the mutated cDNA was restricted
with BamHI/SacI and cloned in the pBI121 vector pre-
viously restricted with the same enzymes.
Isolation and cloning of ATHB1 cDNA
Arabidopsis plants (Col 0) were germinated and grown in
MS-agar medium during 14 days and after that placed in
liquid MS (5 ml; 35 mm diameter vessels) supplemented
with 300 mM mannitol during additional 24 h. After
that, RNA was isolated by the Trizol method (Invitrogen,
Carlsbad, CA, USA), following the manufacturer instruc-
tions, as previously described [26] (RT reactions were
performed with 1 μgRNAand200unitsofM-MLV
(Promega). PCR on cDNA was performed using oligonu-
cleotides ATHB1F and ATHB1R for A THB1, ATHB1F
and ATHB1WCTR for ATHB1WCT, and ATH1CTF
and ATHB1R for ATHB1CT (Table 3), and the PCR
Table 3 Oligonucletotides used for cloning
Oligonucleotide name Sequence Restriction sites construct

Transfl 5’-gC
ggATCCACCATgTCTTTCAACAAgTA-3’ BamHI H4-H1 cloning
H1CDS-R 5’-ggg
gAgCTCTCAATTgAATTgTggTTgTTCC-3’ SacI H4-H1 cloning
H1*H4F 5’-gTTggAggTgTAAAAAATAgggAgCCAgC-3’ H4-H1 cloning
H1*H4R 5’-TATTTTTTTAgCACCTCCAACTgATTgAgTAgg-3’ H4-H1 cloning
H4-WCT-R 5’-CCC
gAgTCTCTATTCTTCACCgCTgCCAC-3’ SacI H4WCT cloning
H1-WCT-R 5’-ggC
gAgCTCTCATCCTTCTgTTTCTTTTATgTTgAgg-3’ SacI H1WCT cloning
H1atF 5’-ggg
ggATCCgCTgATgACTTgCACTggAATggC-3’ BamHI H1WCT cloning
H1qR 5’-CCAACCATggCCAAAACCCTg-3’ H1 qRT-PCR
H1qF 5’-ggCCggCAgATCATCAACTTC-3’ H1 qRT-PCR
H4qR 5’-gCCgAgTCTTAgAACTCCAACCACTTTTg-3’ H4 qRT-PCR
H4qF 5’-CgCgATCAAAgTCgAggCAgATTg-3’ H4 qRT-PCR
H4quimqF 5’-CgCgATCAAAgTCgAggCAgATTg-3’ H4-H1 qRT-PCR
H1AsTH4R 5’-TCCTTCTgTTTCTTTTATgTTgAgg-3’ H4-H1 qRT-PCR
UBCQPCR-R 5’-CAgTggACTCgTACTTgTTCTTgT-3’ Genomic DNA
UBC9GENOM-F 5’-gTTTTggAAATgTTgACAggAC-3’ Genomic DNA
ATHB1F 5’ gCg
gAATTCATggAATCCAATTCgTTTTTC 3’ EcoRI ATHB1 and ATHB1WCT cloning
ATHB1R 5’ gCg
ggATCCTAAggCCATCCCCAgAAAg 3’ BamH1 ATHB1 cloning
ATHB1WCTR 5’ gCg
gTCgACTACTCTTgTTTgCCCTgAAgC 3’ Sal1 ATHB1WCT cloning
ATHB1CTF 5’ gCggAATTCCAAgAgACAgCTAATgAACCA 3’ EcoRI ATHB1 CTR cloning
Sequence of oligonucleotides used in PCR reactions to perform constructs or cDNA quantifying. In bold, the overlapping sequences for chimerical constructs and
underlined the restriction sites used for cloning.
Arce et al. BMC Plant Biology 2011, 11:42

/>Page 15 of 19
products cloned into the EcoRI/Bam HI or the EcoRI/Sal
I sites of the pGBKT7 vector, respectively.
Yeast culture and transformation
Saccharomyces cerevisiae AH109 (Clontech) cells were
grown in YPDA or synthetic minimal medium (SD) sup-
plemented with an amino acids dropout solution defi-
cient in Trp or His [74]. Yeast cells were transformed
with the previously obtained GAL4-ATHB1, GAL4-
ATHB1WCT and GAL4-ATHB1CT chimerical con-
structs in pGBKT7 following the lithium acetate method
[75]. Transformed cells were selected for tryptophan
prototrophy on SD medium.
Transcriptional activation ability was assayed by
b-galactosidase colony lift filter as suggested by the
manufacturer (Clontech).
Plant material and growth conditions
Arabidopsis thaliana Heyhn. Ecotype Columbia (Col-0)
was purchased from Lehle Seeds (tucson, AZ). Plants
were grown directly on soil in a growth chamber at 22-
24 °C under long-day photoperiods (16 h of illumination
with a mixture of cool-white and GroLux fluorescent
lamps) at intensity of approximately 150 μEm
-2
s
-1
in
8 cm diameter × 7 cm height pots.
Plant transformation
Transformed Agrobacterium tumefaciens strain

LBA4404 was used to obtain transgenic Arabidopsis
plants by the floral dip procedure [76]. Transformed
plants were selected on the basis of kanamycin resis-
tance and positive PCR which was carried out on geno-
mic DNA with specific oligonucleotides for each
construct as indicated in Table 3. Fifteen positive in de-
pendent lines for each construction were used to select
homozygous t3 and t4 plants in order to analyze pheno-
types. Plants transformed with pBI101.3 were used as
negative controls.
Real time RT-PCR measurements
Expression levels of each transcri pt in transgenic plants
were quantified by qPCR as follows. RNA was prep ared
with Trizol
®
reagent (Invitrogen™) according to the
manufacturer’s instructions. RNA (2 μg) was used for
the RT reactions using M-MLV reverse transcriptase
(Promega). Quantitative PCRs were carried out using a
MJ-Chromo 4 apparatus in a 20 μl final volume contain-
ing 1 μl SyBr green (10 ×), 8 pmol of each primer, 2
mM Mgcl
2
,10μl of a 1/25 dilution of the Rt reaction
and 0,12 μl Platinum Taq (Invitrogen Inc.). Fluorescence
was measured at 78-80°C during 40 cycles. Specific oli-
gonucleotides for each gene were designed and their
sequences specified in Table 3.
Ethylene treatments
Seeds were surface-sterilized and plated with MS med-

ium-0,8% agar in Petri dishes. After 2 days of incubation
at 4°CC, dishes were placed in a growth chamber at 22-
24°C. Dark grown seedlings were grown on 5 μM ACC,
and maintained during the same period of time.
Quantification of leaf serration
Leaves from 21-day-old plants w ere excised and images
were acquired with a regular fla tbed image scanner.
These images were processed with the program
LAMINA [77] which is designed to recognize different
shape parameters of leaves, including serration. The
number of serrations per leaf was calculated in WT,
HAHB1 B, HAHB4 B and H4-H1 A, B and C plants.
The results (Figure 8) showed that HAHB1 and H4-H1
B plants presented a clear increase in serration while the
rest of the lines had a serration similar to WT plants.
These results were subjected to the Kruskal-Wallis one-
way analysis of variance by ranks and then the different
lines were classified in groups according to pairwise
comparisons with a p-value of 0,05 (Table 2).
Additional material
Additional file 1: Sequences used in the analysis. This spreadsheet file
contains the sequences of the proteins used in this work, plus additional
information.
Additional file 2: Sequence alignment of the HD-Zip domains. The
HD-Zip domains of the 178 proteins plus the three outgroups were
processed for alignment. This alignment was used for the HZT (only the
178 HD-Zip I proteins) and the HZT + OG.
Additional file 3: Sequence alignment of the complete proteins. The
complete sequences of the 178 proteins were aligned for the
construction of the CST.

Additional file 4: HZT and CST. The phylogenetic trees are shown with
their complete topology.
Additional file 5: HZT + OG. Phylogenetic tree used to root the HZT
and CST.
Additional file 6: Alignment of the CTRs. The CTRs of the proteins of
each group were aligned showing the conservation in this region.
Additional file 7: CTR putative phosphorylation sites. Each of the
CTRs is represented by three stacked sequences: i) only the residues
predicted to be phosphorylated are visible, the remaining are substituted
with dots; ii) the visible residues correspond to motifs found by the
MEME program; and iii) the complete sequence is visible.
Additional file 8: Motif distribution in the NTRs. In an analogous
representation to the one in Figure 4, the distribution of motifs in the
NTRs is depicted for each protein. The tree on the left represents their
phylogenetic relationships. The analysis is divided in three separate plots
and the groups identified previously (i.e., I-VI) are highlighted with boxes
of dashed boundaries. Putative phosphorylation sites (Ser, Thr, Tyr) are
marked with a black diamond, sumoylation motifs with a blue inverted
triangle and NLSs with green crosses.
Additional file 9: Motifs found in the NTRs. The sequence logos of
the motifs found in the NTRs by the program MEME are displayed.
Additional file 10: NTRs putative phosphorylation sites. Each of the
NTRs is represented by three stacked sequences: i) only the residues
predicted to be phosphorylated are visible, the remaining are substituted
Arce et al. BMC Plant Biology 2011, 11:42
/>Page 16 of 19
with dots; ii) the visible residues correspond to motifs found by the
MEME program; and iii) the complete sequence is visible.
Additional file 11: NLStradamus report. Text file with the report of the
analysis performed with the program NLStradamus on the complete

sequences.
Abbreviations
ABA: abscicic acid; ACC: 1-aminocyclopropane-1-carboxylic-acid; AHA:
Aromatic, large Hydrophobic, Acidic context; CST: complete sequence tree;
CTR: carboxy-terminal region; ET: ethylene; HALZ: homeodomain-associated
leucine zipper; HD-Zip: homeodomain-leucine zipper; HSF: Heat Stress
Transcription Factors; JA: jasmonic acid; NTR: amino-terminal region; SAGA:
Spt-Ada-Gcm5-Acetyltrasnferase; SWI/SNF: SWItch/Sucrose Non Fermentable;
TF: transcription factor; TFIID: transcription factor IID; TL: Tendril-less
Acknowledgements
We acknowledge the staff at “Centro Internacional de Métodos
Computacionales en Ingeniería” (CIMEC, , CONICET
and UN Litoral) for granting access to their computing cluster Aquiles
(grants FONCyT PME 209, PICT 1141/2007, PICT 1506/2006); and in particular
to Dr. Mario Storti for his helpful assistance.
This work was supported by ANPCyT (PICT 2008 1206 and PICT-PAE 37100),
and UNL. ALA, JVC and MC are Fellows of Conicet-Argentina. JR is a Fellow
of Foncyt (PICT-PAE 37100/022) and RLC is a CONICET Career member.
Authors’ contributions
ALA carried out the phylogenetic analysis, the functional characterization of
CTRs and NTRs, analyzed the data and did the illustrations. JR performed the
chimerical constructs, obtained the transgenic plants and did the triple
response experiments. MC performed the cloning of ATHB1 in its three
versions and did the yeast transactivation assays. JC together with ALA and
JR analyzed the phenotype of the transgenic plants. RLC conceived this
study, participated in the design and coordination and together with ALA
drafted the MS. All authors read and approved the final manuscript.
Received: 16 November 2010 Accepted: 3 March 2011
Published: 3 March 2011
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Cite this article as: Arce et al.: Uncharacterized conserved motifs outside
the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential
source of functional diversity. BMC Plant Biology 2011 11:42.
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